Chlorothalonil exposure induces “liver-gut axis” disorder in mice

Chlorothalonil (CTL) is a broad spectrum, non-systemic, organo-chlorine fungicide, widely used in agriculture, silviculture, urban settings, and industrial antifouling. It is found in various environmental media, such as surface water, soil, and air, and even ex-ceeded Maximum residual limit (MRL) in food chain [1,2]. Previous studies revealed that CTL is highly toxic to aquatic organisms and amphibian, especially in the early stage of development [3]. Under experimental conditions, it may also have toxic effects on rodents, mammalian cells, and other non target organisms. In humans, the main exposure modes of CTL were contacting to the residues in the work/life place or intaking the contaminated dietary, which may cause contact allergic dermatitis, occupational asthma, gastro-intestinal problems and other symptoms [4]. However, the effects of CTL on the “liver-gut axis” have not been reported yet. In this study, after one week of adaptation to the environment, six-week-old male ICR mice (China

Mix (Vazyme, Beijing, China) and primers listed in Supplementary  Table S2. As shown in Figure 1A, CTL exposure notably downregulated the expressions of genes related to glycolipid metabolism. For example, pruvate kinase (PK) and carbohydrate regulatory element binding protein (Chrebp), which are responsible for glycolysis, showed a down-regulated trend in CTL-treated groups when compared with the control group. Their down-regulation could raise the GLU storage and reduce hepatic pyruvate level in the liver. However the change of transcriptional level of glucokinase (GK) is not clear. The genes involved in fatty acid β-oxidation, such as peroxisome proliferator-activated receptor α (PPAR-α), Cpt1a and ACOX, all showed a down-regulated trend, especially in the CTL-800 group ( Figure 1B). CTL exposure also reduced the expressions of genes participated in fatty acid synthesis and transportin the liver, such as PPAR-γ, citrate lyase (Acl), stearoyl COA desaturase 1 (Scd1) and fatty acid binding protein (Fabp1) ( Figure  1C,D). The mRNA levels of genes related to TG synthesis, including diacylglycerol acyltransferase 2 (Dgat2) and glyceraldehyde 3phosphate acyltransferase (Gpat) were also declined in the CTL-800group ( Figure 1E). Maybe it is a stress response to hyperlipidemia in the liver. These results suggested that CTL exposure reduced the ability of glycolipid metabolism in the liver through downregulating thetranscriptional levels of key genes.
We further analyzed hepatic metabolites by liquid chromatography tandemmass spectrometry (LC-MS/MS) between the control group and CTL-800 group. Liver tissue extraction was performed as previously reported [5]. Through principle component analysis (PCA), we found that the patterns of metabolites were significantly different from both positive and negative between the control and CTL-800 group ( Figure 1G,I). Among of the high-quality feature, a total of 371 metabolites were increased and 402 metabolites were decreased in the positive-model ( Figure 1F). Correspondingly, a total of 320 metabolites were increased and 341 metabolites were decreased in negative-model ( Figure 1H). We screened the metabolites (|log2(Fold Change)|≥1, VIP ≥1, Q value≤0.05) obtained from MS2 according to the HMDB database and observed significant

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Liver-gut axis disorder in mice changes in benzenoids, organic acids and derivatives, organ oheterocyclic compounds, lipids and lipid-like molecules, organic oxygen compounds, organooxygen compounds, nucleosides, nucleotides, and analogues between the control group and CTL-800 group ( Figure 1J). Among these, the contents of deoxycholic acid, linoleic acid, tauro-b-muricholic acid and taurocholic acid which are related to bile acids (BAs) metabolism were found to have different degrees of changes after exposure to 800 μg/L CTL, indicating that BA metabolism was also disrupted.
We further detected the transcriptional levels of genes responsible for BAs synthesis, transportation and the signal molecular of BAs hepatointestinal circulatory and found that the expression of cyp7a1, a rate-limiting enzyme of BAs synthesis, was significantly suppressed at the transcriptional level in the CTL-800 treated group (Figure 2A). MRP3, which is related to BAs secretion into systemic circulation, was significantly up-regulated in all CTL treatment groups.
Meanwhile, genes regulating BAs re-absorption in the ileum were equally perturbed by CTL exposure (Figure 2A,B). Abst, Ostα and Ostβ were significantly down-regulated in high dosage group, and both Ostα and Ostβ were decreased even in low dosage group. Farnesoid X receptor (FXR) is a member of the nuclear receptor family regulating the expression of Fgf15, which regulates the negative feedback of BA synthesis [6]. It was decreased significantly in all CTL exposure groups in the ileum and in the liver of the CTL-400 group (Figure 2A). In addition, all the possible roles of these genes in the pathways in liver-intestine circulation metabolism of BAs are shown in Figure 2A.
The live and gut have the same embryological origin. They have a natural and extensive connection in structure and function. The intestinal blood reflux forms the portal vein system into the liver. Intestinal toxins require the liver to rely on its innate immune system to play a defensive role. The liver regulates metabolism and immune response, and affects intestinal function through bile secretion and enterohepatic circulation. Liver disease can lead to the weakening of immune defense mechanism of intestinal barrier and the damage of tight junction of epithelial cells [1]. Their pathophysiological relationship is expressed as "liver-gut axis". The imbalance of BAs metabolism will also affect the physiological function between liver and intestine [7]. Our previous studies confirmed that CTL exposure will increase intestinal cell apoptosis and destroy the integrity of intestinal barrier on Caco-2 monolayer model [8,9].
Based on this finding, we detected the related indexes of intestinal barrier integrity by western blot analysis of the colon samples collected as previously described [9] using antibodies listed in Supplementary Table S3. We found that the expressions of tight junctions (TJs) proteins Zo-1 and Cldn 1 had downward trends, and the expressions of Erk1/2 and Jnk, two key members in MAPK signaling pathway in the regulation of intestinal epithelium barrier stability, were also declined ( Figure 2C,D). Quantitative PCR revealed that the expressions of TJ genes, including Zo-1, Cldn 1 and Ocln, had down-regulated trend, which is the same as that of the Sis (Brush border marker) gene, and the change of Muc2 is significant ( Figure 2E). At the same, the expressions of apoptosis-related genes, Bax and Caspase 3 showed up-regulated trend, the change of Bad is significant, andexpression of Bcl-2 was accordingly decreased, though it is not significant ( Figure 2F).
In summary, in this study we observed that sub-chronic exposure to CTL disturbed "liver-gut axis" metabolism, mainly including

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Liver-gut axis disorder in mice glucose and lipid metabolism and intestinal barrier integrity, at the transcription and protein expression level. In addition, the metabolism of BAs which are one of the bridges connecting the liver and gut is also influenced. Based on the results, we speculate that CTL may have toxic effects on liver metabolism through disturbing BAs metabolism. With regard to the "liver-gut axis", the damage of the intestinal barrier may also be involved in the functional abnormalities of liver metabolism.

Supplementary Data
Supplementary data is available at Acta Biochimica et Biophysica Sinica online.

Funding
This work was supported the grant from the Zhejiang Provincial Natural Science Foundation of China (No. LZ20B070002).